Endotoxin binding lactic acid bacteria and bifidobacteria

ABSTRACT

The invention relates to the use of at least one strain of lactic acid bacteria and/or bifidobacteria having hydrophobic surface properties, for the preparation of a composition intended for the prevention or the treatment of endotoxin mediated and/or associated disorders and human or pet food compositions prepared thereof.

[0001] The present invention relates to the use of lactic acid bacteriaand/or bifidobacteria that are hydrophobic for the preparation of a foodcomposition intended for the prevention or the treatment of endotoxinmediated and/or associated disorders. The invention also relates tocomposition prepared thereof.

BACKGROUND OF THE INVENTION

[0002] Sepsis-inducing toxins have been found associated with pathogenicbacteria, viruses, plants and venom. Among the well-described bacterialtoxins are the endotoxins or lipopolysaccharides (LPS) of thegram-negative bacteria. These molecules are glycolipids that areubiquitous in the outer membrane of all gram-negative bacteria, whichare believed responsible for gram-negative sepsis. This type of sepsisis an extremely common condition and is often fatal.

[0003] A number of approaches for treating sepsis have been attempted.These include use of antibodies to LPS, use of antibodies to tumournecrosis factor, use of a soluble TNF receptor, use of a solubleinterleukin-1 (IL-1) receptor, to name a few. While each approach hassome efficacy, the overall results have been disappointing.

[0004] Others have attempted to design and study proteins which bindLPS/endotoxin, and illustrative reports of these attempts appear inRustici, A. et al. Science (1993) 259:361-364; Matsuzaki, K. et al.Biochemistry (1993) 32:11704-11710; Hoess, A. et al. EMBO J (1993)12:3351-3356; and Elsbach, P. et al. Current Opinion in Immunology(1993) 5:103-107. In fact, upon introduction of LPS into the blood, itmay bind to a protein termed lipopolysaccharide-binding protein (LBP).Inhibition of LBP, e.g., with an anti-LBP antibody, has been suggestedas therapeutically useful for treating endotoxin-mediated sepsis(International Patent Application No. PCT/US90/04250, filed Jul. 30,1990). Also, work from several laboratories has shown that plasmalipoproteins, particularly high-density lipoproteins (HDL), bind andneutralise LPS (Skarnes et al., 1968, J. Bacteriology 95:2031; Flegel etal., 1993, Infect. Immunol. 61(12):5140) and that these particles mayconstitute the LPS-neutralising activity in plasma.

[0005] Previous treatments for endotoxin mediated and/or associateddiseases has been retrospective (i.e., after development of clinicalillness) and has been limited to chemotherapeutic intervention.Prevention measures were not achieved with such treatments.

[0006] Thus, there is a need in the art for an effective agent forneutralising gram-negative endotoxin (i.e., LPS), in order to prevent oralleviate symptoms of sepsis and septic shock.

[0007] The hydrophobic lactic acid bacteria and bifidobacteria of thepresent invention provide additional compounds which are capable ofbinding endotoxins and ameliorating/preventing its effects.

SUMMARY OF THE INVENTION

[0008] Thus, the present invention relates to the use of at least onestrain of lactic acid bacteria and/or bifidobacteria that hashydrophobic surface properties, for the preparation of a compositionintended for the prevention or the treatment of endotoxin mediatedand/or associated disorders.

[0009] In fact, it has been surprisingly found that some lactic acidbacteria and bifidobacteria, particularly those with hydrophobicsurface, have the ability to bind endotoxins. Thus, allowing their useas efficient agents for prevention of endotoxic shock and sepsis of gutorigin, bacterial translocation, necrotising enterocolitis, inflammatorybowel disease, intestinal infections, chronic endotoxemia associated orpromoting catabolic and systemic inflammation.

[0010] Preferably, the hydrophobic lactic acid bacteria orbifidobacteria has a percent hydrophobicity (% H) of at least 80, andmore preferably from 85 to 100% H.

[0011] In a preferred embodiment, the strain is selected from the groupconsisting of Lactobacillus johnsonii, Lactobacillus reuterii,Lactobacillus paracasei, Lactobacillus animalis, Lactobacillus ruminis,Lactobacillus acidophilus, Lactobacillus rahmnosus, Lactobacillusfermentum, Lactobacillus delbrueckii subs. lactis, Bifidobacterium spp.,Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacteriumpseudolongum, Bifidobacterium infantis, Bifidobacterium adolescentis.

[0012] In another aspect, the invention relates to an isolated strain oflactic acid bacteria or bifidobacteria having hydrophobic surfaceproperties, that has been selected for its ability to bind endotoxins orco-aggregate with gram-negative bacteria.

[0013] In a further aspect, the invention provides a human or pet foodcomposition for preventing or treating endotoxin mediated and/orassociated disorders, which contains at least a strain of lactic acidbacteria and/or bifidobacteria having the above traits, associated withan ingestible support or a pharmaceutical matrix.

[0014] This composition presents the advantage to decrease small bowelbacterial overgrowth and diminish the endotoxin leakage from the gut tothe internal milieu, a frequent disorder found in pets that may causeepisodes of diarrhoea, malnutrition and intestinal and systemicinflammation, for example.

[0015] In a last aspect, the invention relates to a method of preventingor treating endotoxin mediated and/or associated disorders, comprisingthe step of feeding a human or animal a composition which contains atleast a strain of lactic acid bacteria and/or bifidobacteria that hashydrophobic surface properties, associated with an ingestible support ora pharmaceutical matrix

DETAILED DESCRIPTION OF THE INVENTION

[0016] Within the following description, “NCC” designates Nestlé CultureCollection (Nestlé Research Centre, Vers-chez-les-Blanc, Lausanne,Switzerland).

[0017] With respect to the first object of the present invention, theuse of at least one strain of lactic acid bacteria and/or bifidobacteriathat has hydrophobic surface properties, for the preparation of acomposition intended for the prevention or the treatment of endotoxinmediated and/or associated disorders, is concerned.

[0018] In fact, it has been surprisingly found that some lactic acidbacteria and bifidobacteria, particularly those with hydrophobicsurface, have the ability to bind endotoxins.

[0019] Indeed, the bacterial strain according to the invention has theability to bind endotoxins on the hydrophobic cell wall and thereforescavenge this proinflammatory product of gram negative bacteria thatotherwise may translocate from the lumen of the gut into the blood andthereby trigger inflammatory reactions and, in very serious cases,endotoxic shock.

[0020] The lactic acid bacteria or bifidobacteria according to theinvention have been selected among strains suitable for animalconsumption, with regard to their percent hydrophobicity (% H), asdescribed in A. G. Zavaglia et al., Journal of Food protection, Vol. 61,No.7, 1998, p.865-873.

[0021] Preferably, the bacterial strain according to the invention has a% H of at least 80, and more preferably from 85 to 100% H.

[0022] The determination of surface hydrophobicity was done by using theMATH method as previously described (Pérez, P. F. et al., 1998, Appl.Environ. Microbiol. 64: 21-26). Briefly, 2 ml of bacterial suspension(around 10⁸ CFU/ml, PBS) were extracted with 0.4 ml of xylene byvortexing them during 120 s. The phases were allowed to separe bydecantation and A₆₀₀ of the aqueous phase was measured. Cell surfacehydrophobicity (% H) was calculated with the formula H%=[(A₀−A)/A₀]×100where A₀ and A are absorbances before and after extraction with xylenerespectively.

[0023] In a preferred embodiment the bacterial strain may be selectedfrom the group consisting of Lactobacillus johnsonii, Lactobacillusreuterii, Lactobacillus paracasei, Lactobacillus animalis, Lactobacillusruminis, Lactobacillus acidophilus, Lactobacillus rahmnosus,Lactobacillus fermentum, Lactobacillus delbrueckii subs. lactisBifidobacterium spp., Bifidobacterium bifidum, Bifidobacterium longum,Bifidobacterium pseudolongum, Bifidobacterium infantis, Bifidobacteriumadolescentis.

[0024] In a most preferred embodiment the strain may be Lactobacillusacidophilus NCC 2463 (CNCM-I 2623), Bifidobacterium bifidum NCC 189(previously CIDCA 536, CNCM-I-2333), Bifidobacterium bifidum NCC 235(previously CIDCA 533, CNCM-I-2335), Bifidobacterium adolescentis NCC251 (CNCM 1-2168), Bifidobacterium lactis (ATCC27536), for example.

[0025] Among the various strains selected in accordance with the presentinvention, the following strains were deposited by way of example underthe Budapest Treaty at the Collection Nationale de Cultures deMicroorganismes (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724Paris Cedex 15, France, Lactobacillus acidophilus NCC 2463 on the2.02.2001 under the reference CNCM 1-2623, and Bifidobacterium bifidumNCC 189 and NCC 235 on the 12.10.99 under the references CNCM-I-2333 andCNCM 1-2335, respectively. Bidifobacterium adolescentis NCC 251 wasdeposited on the 15.03.99 under the reference CNCM 1-2168.

[0026] The strain of Bifidobacterium lactis (Bb12) (ATCC27536) isprovided by Hansen (Chr. Hansen A/S, 10-12 Boege Alle, P.O. Box 407,DK-2970 Hoersholm, Danemark). It has a hydrophobicity of 89% H.

[0027] The bacterial strain according to the present invention may beused for the preparation of compositions intended for improving human oranimal health, particularly for the prevention or treatment of disordersrelated to endotoxins in humans and pets. The bacterial strain may beused as efficient agent for prevention of endotoxic shock and sepsis ofgut origin, bacterial translocation, necrotising enterocolitis,inflammatory bowel disease, intestinal infections, and chronicendotoxemia associated or promoting catabolic and systemic inflammation,for example.

[0028] The bacterial strain according to the invention may be used inits viable or inactivated form.

[0029] In a preferred embodiment the lactic bacteria strain is used inthe presence of its fermented growth medium. The said medium can beeither sterilised alone or with a food, extruded or spray-dried, chilledor shelf stable, for example.

[0030] The bacterial strain may be used so that the amount available forthe individual may correspond to about 10³-10¹⁴ cfu per day. This amountdepends on the individual weight, and it is preferably of about 10⁹-10¹²cfu/day for humans and 10⁷-10¹⁰ cfu/day for pets.

[0031] According to another aspect, the present invention relates to anisolated strain of lactic acid bacteria or bifidobacteria havinghydrophobic surface properties and having the ability to bind endotoxinsor co-aggregate with gram-negative bacteria.

[0032] The ability of the bacterial strain according to the presentinvention to bind endotoxins can be measured easily using FITC-labelledendotoxins, measuring the association of radiolabelled endotoxin tobacterial cells, in this case the molecular structure of the endotoxinis unaltered as compared with the possible modifications that may sufferthe molecule after conjugation with the FITC (see examples).

[0033] Preferably, the capacity of the bacterial strain to removeendotoxin from a solution that mimics the quantity of endotoxins foundin the human intestine is measured. For example, levels of endotoxinswere examined with the microassay for the detection of2-keto-3-deoxyoctonate group in the lipopolysaccharide of gram-negativebacteria (Karkhanis Y D, et al., Analytical Chemistry (1978) 85:595-601). Bacteria removing more that 30% of the endotxin content fromthese solutions were preferably selected. The non-hydrophobic bacteriatested in this assay were unable to modify the initial content ofendotoxin (see examples).

[0034] Such bacterial strain may be used as described above, andparticularly as efficient agent for prevention of endotoxic shock andsepsis of gut origin, bacterial translocation, necrotisingenterocolitis, inflammatory bowel disease, intestinal infections,chronic endotoxemia associated or promoting catabolic and systemicinflammation, for example.

[0035] According to a further aspect, the present invention relates to ahuman or pet food composition containing at least an isolated strain oflactic acid bacteria and/or bifidobacteria, said strain having the abovetraits, associated with an ingestible support or a pharmaceuticalmatrix.

[0036] The strain may be selected as described above.

[0037] Preferably, the lactic acid bacteria or bifidobacteria may beadministered as a supplement to the normal diet or as a component of anutritionally complete human or pet food.

[0038] The human or pet food composition may comprise at least thelactic acid bacteria and/or bifidobacteria strain, as described above,so that the amount available for the individual may correspond to about10³-10¹⁴ cfu per day. This amount depends on the individual weight, andit is preferably of about 10⁹-10¹² cfu/day for humans and 10⁷-10¹⁰cfu/day for pets.

[0039] The human food may be in the form of a nutritional formula, aninfant formula, milk-based products, dairy products, cereal-basedproducts, for example. To prepare such a food product or composition,the bacterial strain as described above can be incorporated into a food,such as cereal powder, milk powder, a yoghurt, during its manufacture,for example.

[0040] In one embodiment, a nutritional formula comprising a source ofprotein and at least one bacterial strain according to the invention canbe prepared. Dietary proteins are preferably used as a source ofprotein. The dietary proteins may be any suitable dietary protein; forexample animal proteins (such as milk proteins, meat proteins and eggproteins), vegetable proteins (such as soy, wheat, rice or peaproteins), mixtures of free amino acids, or combination thereof. Milkproteins such as casein, whey proteins and soy proteins are particularlypreferred. The composition may also contain a source of carbohydratesand a source of fat.

[0041] If the nutritional formula includes a fat source, the fat sourcepreferably provides about 5% to about 55% of the energy of thenutritional formula; for example about 20% to about 50% of the energy.The lipids making up the fat source may be any suitable fat or fatmixtures. Vegetable fats are particularly suitable; for example soy oil,palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canolaoil, lecithin, and the like. Animal fats such as milk fats may also beadded if desired.

[0042] If the nutritional formula includes a carbohydrate source, thecarbohydrate source preferably provides about 40% to about 80% of theenergy of the nutritional formula. Any suitable carbohydrates may beused, for example sucrose, lactose, glucose, fructose, corn syrupsolids, and maltodextrins, and mixtures thereof.

[0043] Dietary fibre may also be added if desired. Numerous types ofdietary fibre are available. Suitable sources of dietary fibre, amongothers, may include soy, pea, oat, pectin, guar gum, and gum arabic. Ifused, the dietary fibre preferably comprises up to about 5% of theenergy of the nutritional formula. Suitable vitamins and minerals may beincluded in the nutritional formula in the usual manner to meet theappropriate guidelines. One or more food grade emulsifiers may beincorporated into the nutritional formula if desired; for examplediacetyl tartaric acid esters of mono-diglicerides, lecithin and mono-and di-glycerides. Similarly suitable salts and stabilisers may beincluded.

[0044] The nutritional formula is preferably enterally administrable;for example in the form of a powder, a liquid concentrate, or aready-to-drink beverage.

[0045] The nutritional formula may be prepared in any suitable manner.For example, the nutritional formula may be prepared by blendingtogether the source of dietary protein, the carbohydrate source, and thefat source in appropriate proportions. If used, the emulsifiers may beincluded in the blend. The vitamins and minerals may be added at thispoint but are usually added later to avoid thermal degradation. Anylipophilic vitamins, emulsifiers and the like may be dissolved into thefat source prior to blending. Water, preferably water that has beensubjected to reverse osmosis, may then be mixed in to form a liquidmixture. The temperature of the water is conveniently about 50° C. toabout 80° C. to aid dispersal of the ingredients. Commercially availableliquefiers may be used to form the liquid mixture. The liquid mixture isthen homogenised; for example in two stages.

[0046] The liquid mixture may then be thermally treated to reducebacterial loads. For example, the liquid mixture may be rapidly heatedto a temperature in the range of about 80° C. to about 150° C. for about5 seconds to about 5 minutes. This may be carried out by steaminjection, autoclave or by heat exchanger; for example a plate heatexchanger. The liquid mixture may then be cooled to about 60° C. toabout 85° C.; for example by flash cooling. The liquid mixture may thenbe again homogenised; for example in two stages at about 7 MPa to about40 MPa in the first stage and about 2 MPa to about 14 MPa in the secondstage. The homogenised mixture may then be further cooled to add anyheat sensitive components; such as vitamins and minerals. The pH andsolids content of the homogenised mixture is conveniently standardisedat this point.

[0047] If it is desired to produce a powdered nutritional formula, thehomogenised mixture is transferred to a suitable drying apparatus suchas a spray drier or freeze drier and converted to powder. The powdershould have a moisture content of less than about 5% by weight.

[0048] If it is desired to produce a liquid formula, the homogenisedmixture is preferably aseptically filled into suitable containers.Aseptic filling of the containers may be carried out by pre-heating thehomogenised mixture (for example to about 75 to 85° C.) and theninjecting steam into the homogenised mixture to raise the temperature toabout 140 to 160° C.; for example at about 150° C. The homogenisedmixture may then be cooled, for example by flash cooling, to atemperature of about 75 to 85° C. The homogenised mixture may then behomogenised, further cooled to about room temperature and filled intocontainers. Suitable apparatus for carrying out aseptic filling of thisnature is commercially available. The liquid formula may be in the formof a ready to feed formula having a solids content of about 10 to about14% by weight or may be in the form of a concentrate; usually of solidscontent of about 20 to about 26% by weight. Flavours may be added to theliquid formulas so that the formulas are provided in the form ofconvenient, flavoursome, ready-to-drink beverages.

[0049] In an another embodiment, a usual food product may be enrichedwith the bacterial strain according to the present invention. Forexample, a fermented milk, yoghurt, a fresh cheese, a renneted milk, aconfectionery bar, breakfast cereal flakes or bars, drinks, milkpowders, soy-based products, non-milk fermented products or nutritionalsupplements for clinical nutrition.

[0050] In a further embodiment, a nutritionally complete pet foodcomposition can be prepared. It may be in powdered, dried form,semi-moist or a wet, chilled or shelf stable pet food product. It canalso be dietary supplements for pets or pharmaceutical compositions.These pet foods may be produced as is conventional. Apart from thebacteria strain, these pet foods may include any one or more of a starchsource, a protein source and lipid source.

[0051] Suitable starch sources are, for example, grains and legumes suchas corn, rice, wheat, barley, oats, soy, and mixtures of these. Suitableprotein sources may be selected from any suitable animal or vegetableprotein source; for example meat and meal, poultry meal, fish meal, soyprotein concentrates, milk proteins, gluten, and the like. For elderlyanimals, it is preferred for the protein source to contain a highquality protein. Suitable lipid sources include meats, animal fats andvegetable fats. Further, various other ingredients, for example, sugar,salt, spices, seasonings, vitamins, minerals, flavouring agents, fatsand the like may also be incorporated into the pet food as desired.

[0052] For dried pet foods a suitable process is extrusion cooking,although baking and other suitable processes may be used. When extrusioncooked, the dried pet food is usually provided in the form of a kibble.If a prebiotic is used, the prebiotic may be admixed with the otheringredients of the dried pet food prior to processing. A suitableprocess is described in European patent application No 0850569; thedisclosure of which is incorporated by reference. If a probioticmicro-organism is used, the organism is best coated onto or filled intothe dried pet food. A suitable process is described in European patentapplication No 0862863; the disclosure of which is incorporated byreference.

[0053] For wet foods, the processes described in U.S. Pat. Nos.4,781,939 and 5,132,137 may be used to produce simulated meat products.The disclosures of these patents are incorporated by reference. Otherprocedures for producing chunk type products may also be used; forexample cooking in a steam oven. Alternatively, loaf type products maybe produced by emulsifying a suitable meat material to produce a meatemulsion, adding a suitable gelling agent, and heating the meat emulsionprior to filling into cans or other containers.

[0054] The amount of prebiotic in the pet food is preferably about 20%by weight; especially about 10% by weight. For example, the prebioticmay comprise about 0.1% to about 5% by weight of the pet food. For petfoods which use chicory as the prebiotic, the chicory may be included tocomprise about 0.5% to about 10% by weight of the feed mixture; morepreferably about 1% to about 5% by weight.

[0055] If a probiotic microorganism is used, the pet food preferablycontains about 10⁴ to about 10¹⁰ cells of the probiotic microorganismper gram of the pet food; more preferably about 10⁶ to about 10⁸ cellsof the probiotic microorganism per gram. The pet food may contain about0.5% to about 20% by weight of the mixture of the probioticmicroorganism; preferably about 1% to about 6% by weight; for exampleabout 3% to about 6% by weight.

[0056] The pet foods may contain other active agents such as long chainfatty acids. Suitable long chain fatty acids include alpha-linoleicacid, gamma linoleic acid, linoleic acid, eicosapentanoic acid, anddocosahexanoic acid. Fish oils are a suitable source of eicosapentanoicacids and docosahexanoic acid. Borage oil, blackcurrent seed oil andevening primrose oil are suitable sources of gamma linoleic acid.Safflower oils, sunflower oils, corn oils and soybean oils are suitablesources of linoleic acid. If necessary, the pet foods are supplementedwith minerals and vitamins so that they are nutritionally complete.

[0057] Further, if desired, the bacteria strain may be encapsulated; forexample in a sugar matrix, fat matrix or polysaccharide matrix.

[0058] The amount of the pet food to be consumed by the pet to obtain abeneficial effect will depend upon the size or the pet, the type of pet,and age of the pet. However an amount of the pet food to provide a dailyamount of about 10³-10¹⁴ cfu of at least one lactic acid bacteria orbifidobacteria strain would usually be adequate. Preferably about10⁹-10¹⁰ cfu/day for dogs or 10⁷-10¹⁰ cfu/day for cats are administered,for example.

[0059] The composition according to the invention is particularlyintended for the prophylaxis or the treatment of infections related togram negative bacteria, endotoxin producing bacteria such asHelicobacter spp, Samonella spp, and also to small intestinal bacterialovergrowth (SIBO) all of which may clinically manifest with diarrhoea,intestinal or systemic inflammatory conditions, or catabolism andmalnutrition.

[0060] According to a last aspect, the invention provides a method ofpreventing or treating endotoxin mediated and/or associated disorders,comprising the step of feeding a human or animal a composition whichcontains at least a strain of lactic acid bacteria and/or bifidobacteriathat has hydrophobic surface properties, associated with an ingestiblesupport or a pharmaceutical matrix.

[0061] This method may be particularly intended for the prophylaxis orthe treatment of infections related to gram negative bacteria, endotoxinproducing bacteria such as Helicobacter spp, Samonella spp, and also tosmall intestinal bacterial overgrowth (SIBO) all of which may clinicallymanifest with diarrhoea, intestinal or systemic inflammatory conditions,or catabolism and malnutrition.

[0062] The following examples are given by way of illustration only andin no way should be construed as limiting the subject matter of thepresent application. All percentages are given by weight unlessotherwise indicated. The examples are preceded by a brief description ofthe figures.

FIGURES

[0063]FIG. 1: Histogram plots for Bifidobacterium bifidum strain NCC 189(previously CIDCA 536, CNCM I-2333) showing binding of FITC-LPS. A:control without FITC-LPS. B: incubation with 50 μl/ml FITC-LPS. At least16000 events were analysed.

[0064]FIG. 2: Effect of albumin on FITC-LPS binding by lactic acidbacteria and bifidobacteria. A: Bifidobacterium bifidum strain NCC 200(previously CIDCA 538, CNCM I-2334). B: Lactobacillus acidophilus strainNCC 2463 (CNCM I-2623). C: Bifidobacterium bifidum strain NCC 189(previously CIDCA 536, CNCM I-2333).

[0065]FIG. 3: Kinetics of growth and FITC-LPS binding forBifidobacterium bifidum strain NCC 189 (previously CIDCA 536, CNCMI-2333). Values are means from two independent experiments.

[0066]FIG. 4: Binding of LPS by Bifidobacterium strain, detected by themicroassay to determine 2-keto-3-deoxyoctonato (KDO) inlipopolysaccharide. The initial concentration of the solution was 100μg/ml. The bars show the final concentration after incubation of thesolution with hydrophobic bifidobacterial strains NCC 189 and NCC 251(CNCM I-2168) compared with a non-hydrophobic strain of bifidobacteria(NCC 200 (CNCM I-2334)).

[0067]FIG. 5: Proinflammatory activity (IL-8 (ng/mL)) of LPS solutionpreincubated with hydrophobic bacteria on HT-29 human epithelial cells.

[0068] Control samples of DMEM, Human milk (HM) 2%, LPS (2.5 μg/ml)alone, Bifidobacterium bifidum strain NCC 189 (1.5e8 cfu/ml) alone weretested for background stimulatory activity. Test solutions of LPS+humanmilk (source of sCD14) were compared with solution containing LPS 2.5μg/ml+HM 2% pre-incubated with Bifidobacterium bifidum strain NCC189(1.5e8 cfu/ml).

EXAMPLES Example 1 Selection of Hydrophobic Lactic Acid Bacteria StrainAccording to the Invention

[0069] The selection of hydrophobic bacteria was initially based in the% of partition of bacterial cells between an organic (hydrophobic) andan aqueous phase. Determination of surface hydrophobicity was done byusing the MATH method as previously described (Pérez, P. F. et al.,1998, Appl. Environ. Microbiol. 64: 21-26). Briefly, 2 ml of bacterialsuspension (around 10⁸ CFU/ml, PBS) were extracted with 0.4 ml of xyleneby vortexing them during 120 s. The phases were allowed to separe bydecantation and A₆₀₀ of the aqueous phase was measured. Cell surfacehydrophobicity (% H) was calculated with the formula H%=[(A₀−A)/A₀]×100where A₀ and A are absorbances before and after extraction with xylenerespectively.

[0070] Subsequently lactic acid bacteria or bifidobacteria were selectedby their capacity to remove endotoxin from a solution that mimics thequantity of endotoxin found in the human intestine. Levels of endotoxinwere examined with the microassay for the detection of2-keto-3-deoxyoctonate group in the lipopolisaccharide of gram-negativebacteria (Karkhanis Y D, et al., Analytical Chemistry (1978)85:595-601).

[0071] Bacteria removing more that 30% of the endotoxin content fromthese solutions were selected. The non-hydrophobic bacteria tested inthis assay were unable to modify the initial content of endotoxin.

Example 2 In-vitro Effect of Lactic Acid Bacteria as Endotoxin Scavenger

[0072] The interaction between LPS from Escherichia coli and lactic acidbacteria bearing different surface properties was studied havingselected bacteria with more of 80% H and some non-hydrophobic “negative”controls the interaction with fluorescent labelled endotoxin wasperformed with flow cytometry.

Materials and Methods

[0073] Bacterial Strains and Growth Conditions

[0074] Strain Lactobacillus acidophilus NCC 2463 (CNCM I-2623) was fromNestec collection (Lausanne, Switzerland). Strains Bifidobacteriumbifidum NCC 189 (previously CIDCA 536, I-2333) and Bifidobacteriumbifidum NCC 200 (previously CJDCA 538, (CNCM I-2334)) were from thecollection of the Centro de Investigacion y Desarrollo en Criotecnologiade Alimentos (La plata, Argentina). Frozen suspensions (−80° C.)preserved with 10% (vol./vol.) glycerol were reactivated once in MRSbroth before experiments. All cultures were conducted at 37° C. inanaerobic conditions (BBL GasPak Plus).

[0075] FITC-LPS Binding

[0076] Lipopolysaccharide and FITC-labelled lypopolysaccharide were fromEscherichia coli serotype 0111:B4 (Skelly, R. R et al., 1979, Infect.Immun. 23: 287-293 and were purchased by Sigma. Stock solutionscontaining 1000 μl/ml were prepared in distilled water and dilutedappropriately.

[0077] Bacterial cultures were washed three times with PBS andsuspensions were standardised to 10⁷ CFU/ml. 400 μl were mixed withdifferent amounts of FITC-LPS or LPS to obtain concentrations rangingfrom 0 to 50 μg/ml in the reaction mixture. Incubations were performedat 4° C. or 37° C. during 30 minutes and then, cells were washed twotimes with PBS and fixed with paraformaldehyde 1% (vol./vol.) during 30minutes at 4° C. Flow cytometric analysis was done using a blue-greenexcitation light (FACScan™).

[0078] Surface Hydrophobicity

[0079] Determination of surface hydrophobicity was done by using theMATH method as previously described (Pérez, P. F. et al., 1998, Appl.Environ. Microbiol. 64: 21-26). Briefly, 2 ml of bacterial suspension(around 10⁸ CFU/ml, PBS) were extracted with 0.4 ml of xylene byvortexing them during 120 s. The phases were allowed to separe bydecantation and A₆₀₀ of the aqueous phase was measured. Cell surfacehydrophobicity (% H) was calculated with the formula H%=[(A₀-A)/A₀]×100where A₀ and A are absorbances before and after extraction with xylenerespectively.

Results

[0080] Surface hydrophobicities of strains under study are shown inTable 1. Values were 93 and 96% for strains Lactobacillus acidophilusNCC 2463 (CNCM I-2623) and Bifidobacterium bifidum NCC 189 (CNCM I-2333)respectively whereas strain Bifidobacterium infantis NCC 200 (CNCMI-2334) was not hydrophobic and showed values around 3%. TABLE 1Hydrophobicities of strains under study. Values are means of at least 3determinations. Strain Hydrophobicity % NCC 2463 (CNCM 1-2623) 93 ± 3NCC 189(CNCM 1-2333) 96 ± 3 NCC 200 (CNCM 1-2334)  3 ± 2

[0081] Incubation of strain NCC 189 with 50 μg/ml of FITC-labelled LPSclearly shifts bacterial population into a fluorescent zone. Eventssituated in the right side of the graph (marker M1) represents 90% ofthe gated population (FIG. 1B). For suspensions incubated withoutFITC-LPS, only 2% of the events are situated in different bindingcapability.

[0082] Table 2 (below) shows that FITC-LPS binding is dose dependent. 5and 15% of FITC (+) cells were found for strains NCC200. On the otherhand, strain NCC 189 shows around 95% of FITC (+) cells. Saturationoccurs around 10 μg/ml of FITC-LPS. Addition of albumin, a lipid binder,clearly reduces FITC (+) ratio in all strains under study (FIG. 2).However, strain CIDCA 536 shows around 30% of FITC (+) cells in thepresence of 0.4% albumin and 50 μg/ml of FITC-LPS (FIG. 2C.).Furthermore for this strain, 10% of FITC (+) cells were found withalbumin concentrations as high as 3%. Divalent cations (Ca2+0.9 mM andMg2+0.5 mM) did not modified binding (data not shown). TABLE 2 Percentof FITC (+) bacteria at different concentrations of FITC-LPS. At least30000 events were analysed. FITC-LPS Strain (μg/ ml) NCC 189 NCC 200 00.1 0.2 1 88.1 0.7 10 92.1 5.2 20 92.8 5.9 30 93.9 3.7 40 94.7 4.6 5094.8 4.6

[0083] Binding of FITC-LPS for Bifidobacterium bifidum strain NCC 189(previously CIDCA 536, CNCM I-2333) was strongly dependent on growthphase (FIG. 3). Bacteria harvested in stationary phase shows a highratio of FITC (+) cells. These values fall dramatically during lag phaseand were progressively restored during growth. No differences betweenbinding at 4° C. and 37° C. were found.

[0084] In summary, highly hydrophobic strains show FITC (+) cells thatreach around 95% for strain NCC 189. Non hydrophobic strain NCC 200never reaches values higher than 5%. These results show that binding ofLPS correlates with surface hydrophobicity.

[0085] These results clearly indicated that hydrophobic bacteria bindFITC-labelled endotoxin and thereby become fluorescent. At aconcentration of 10 μg/ml of endotoxin more than 90% of the hydrophobiccell cultures become fluorescent as detected with flow cytometry; incontrast, less than 10% of non-hydrophobic bacterial cells becamepositive under similar endotoxin concentrations.

[0086] When hydrophobic cultures were introduced in media containingendotoxin at concentration ranging between 30 and 90 μg/ml, bacterialcultures at concentration of 10⁷ to 10⁸ bacterial per ml removed atleast 20% of the present endotoxin. This data was confirmed usingradiolabelled endotoxin.

Example 3 Experiments on Human Immunocompetent Cells

[0087] The abrogation of the pro-inflammatory reaction in humanimmunocompetent cells induced by endotoxin by the presence in theculture system of hydrophobic strains in comparison with non-hydrophobicbacteria was studied.

[0088] Hydrophobic Bifidobacterium bifidum NCC 189 (CNCM I-2333) andnon-hydrophobic Bifidobacterium infantis NCC 200 (CNCM I-2334) wereincubated in a solution containing a defined quantity of endotoxin asdetermined by the KDO method (see example 1) (FIG. 4).

[0089] Endotoxin presence after incubation with bacteria suspension wasclearly diminished by the hydrophobic strain whereas no changes wereobserved for the non-hydrophobic strain.

Example 4 Functional Studies for Hydrophobic Bacteria Materials andMethods

[0090] Washed bacteria were resuspended in DMEM-high glucose (AMIMED)and were preincubated with LPS of Escherichia coli O111: B4 (Sigma) at afinal concentration of 2.5 μg/ml. After centrifugation 200 μl ofsupernatant or resuspended bacterial pellet were used to stimulate HT-29epithelial cells.

[0091] After 20-h culture at 37° C. the HT-29 cell culture supernatantwas examined for the presence of IL-8 using an ELISA technique.

Results

[0092] The results are presented in FIG. 5. Proinflammatory activity ofLPS is significantly reduced when an LPS solution is preincubated withhydrophobic bacteria. After centrifugation the supernatant added to ahuman epithelial cell culture (in the presence of human milk since LPSstimulation is dependent on sCD14 presence) is significantly reducedwith respect to the supernatant that has not been preincubated with thehydrophobic bacteria.

Example 5 Infant Formula

[0093] To obtain an infant formula we prepare the following mixturecontaining for 100 ml of formula: 0.5 to 5%, preferably 2% of peptides,0.2 to 10%, preferably 4% of fat, 1 to 25%, preferably 8% of non-levancarbohydrates (including lactose 65%, maltodextrin 20%, starch 15%), andat least 10⁶ cfu/ml of the following strains: Lactobacillus acidophilusNCC 2463 (CNCM I-2623), Bifidobacterium bifidum NCC189 (CNCM I-2333),Bifidobacterium bifidum CNCM I-2335, Bidifobacterium adolescentis CNCMI-2168, in combination with traces of vitamins and oligoelements to meetdaily requirements, and 0.01 to 2%, preferably 0.3%, of minerals, and 50to 90%, preferably 75% of water.

Example 6 Use of Hydrophobic Lactic Acid Bacteria According to theInvention in Dairy Products

[0094] One or more strain of Bifidobacterium bifidum (CNCM I-2333),Lactobacillus acidophillus NCC 2463 (CNCM I-2623), Bifidobacteriunmbifidum NCC235 (CNCM I-2335) or Bifidobacterium adolescentis NCC251(CNCM I-2168), according to the present invention may be used for themanufacture of fermented yoghurt-like milk products.

[0095] To do this, 11 of a milk product containing 2.8% of fats andsupplemented with 2% of skimmed milk powder and 6% of sucrose isprepared, it is pasteurised at 96° C. for 30 minutes and its temperatureis then lowered to 42° C. Precultures of a non-thickening strain ofStreptococcus thermophilus and of a non-viscous strain Lactobacillusbulgaricus are reactivated in a sterile MSK culture medium containing10% of reconstituted milk powder and 0.1% of commercial yeast extract.

[0096] A preculture of one or more of the strain is also reactivated ina medium containing 10% of reconstituted milk powder and 0.1% ofcommercial yeast extract with 1% sucrose. The pasteurised milk productis then inoculated with 1% of each of these reactivated precultures andthis milk product is then allowed to ferment at 32° C. until the pHreaches a value of 4.5. Fermented milks yoghurt-like products areproduced in this way and stored at 4° C.

Example 7 Dry Dog Food

[0097] A feed mixture is made up of about 58% by weight corn, about 5.5%by weight of corn gluten, about 22% by weight of chicken meal, 2,5%dried chicory, fermented milk by strains of Lactobacillus acidophilusNCC 2463 (CNCM-I 2623)) so that the corresponding amount for the dog isabout 10⁹-10¹⁰ cfu/day, and salts, vitamins and minerals making up theremainder.

[0098] The fed mixture is fed into a preconditioner and moistened. Themoistened feed is then fed into an extruder-cooker and gelatinised. Thegelatinised matrix leaving the extruder is forced through a die andextruded. The extrudate is cut into pieces suitable for feeding to dogs,dried at about 110° C. for about 20 minutes, and cooled to form pellets.

[0099] This dry dog food is able to improve pet health, and particularlyprevents disorders related to endotoxins in pets.

1. The use of at least one strain of lactic acid bacteria and/orbifidobacteria having hydrophobic surface properties, for thepreparation of a composition intended for the prevention or thetreatment of endotoxin mediated and/or associated disorders.
 2. The useaccording to claim 1, wherein the lactic acid bacteria or bifidobacteriahas a percent hydrophobicity (% H) of at least 80, and more preferablyfrom 85 to 100%.
 3. The use according to claim 1 or 2, wherein thelactic acid bacteria and bifidobacteria has the ability to bindendotoxins, or co-aggregate with gram-negative bacteria.
 4. The useaccording to any of claims 1 to 3, wherein the lactic acid bacteriastrain or bifidobacteria is selected from the group consisting ofLactobacillus johnsonii, Lactobacillus reuterii, Lactobacillusparacasei, Lactobacillus animalis, Lactobacillus ruminis, Lactobacillusacidophilus, Lactobacillus rahmnosus, Lactobacillus fermentum,Lactobacillus delbrueckii subs. lactis, Bifidobacterium spp.,Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacteriumpseudolongum, Bifidobacterium infantis, Bifidobacterium adolescentis. 5.The use according to any of claims 1 to 4, wherein the strain isLactobacillus acidophilus NCC 2463 (CNCM-I 2623), Bifidobacteriumbifidum NCC 189 (CNCM-I-2333), Bifidobacterium bifidum NCC 235(CNCM-I-2335), Bifidobacterium adolescentis NCC 251 (CNCM-I-2168),Bifidobacterium lactis (ATCC27536).
 6. The use according to any ofclaims 1 to 5, wherein the lactic acid bacteria is used so that theamount available for the individual is of about 10³-10¹⁴ cfu per day. 7.An isolated strain of lactic acid bacteria or bifidobacteria havinghydrophobic surface properties, that has been selected for its abilityto bind endotoxins or co-aggregate with gram-negative bacteria.
 8. Astrain according to claim 7, wherein the lactic acid bacteria orbifidobacteria binds at least 30% of endotoxin from solutions where theconcentrations are similar to the intestinal content of endotoxin.
 9. Astrain according to claim 7 or 8, selected from the group consisting ofLactobacillus johnsonii, Lactobacillus reuterii, Lactobacillusparacasei, Lactobacillus animalis, Lactobacillus ruminis, Lactobacillusacidophilus, Lactobacillus rahmnosus, Lactobacillus fermentum,Lactobacillus delbrueckii subs. lactis, Bifidobacterium spp.,Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacteriumpseudolongum, Bifidobacterium infantis, Bifidobacterium adolescentis.10. A strain according to any of claims 7 to 9, which is Lactobacillusacidophilus NCC 2463 (CNCM-I 2623), Bifidobacterium bifidum NCC 189(CNCM-I-2333), Bifidobacterium bifidum NCC 235 (CNCM-I-2335),Bifidobacterium adolescentis NCC 251 (CNCM-I-2168), Bifidobacteriumlactis (ATCC27536).
 11. A human or pet food composition containing atleast one strain of lactic bacteria and/or Bifidobacteria according toone of claims 7 to 10, associated with an ingestible support or apharmaceutical matrix.
 12. A composition according to claim 11, whereinthe strain is used in its viable or inactivated form.
 13. A compositionaccording to claim 11 or 12, wherein the lactic acid bacteria strain isselected from the group consisting of Lactobacillus johnsonii,Lactobacillus reuterii, Lactobacillus paracasei, Lactobacillus animalis,Lactobacillus ruminis, Lactobacillus acidophilus, Lactobacillusrahmnosus, Lactobacillus fermentum, Lactobacillus delbrueckii subs.lactis, Bifidobacterium spp., Bifidobacterium bifidum, Bifidobacteriumlongum, Bifidobacterium pseudolongum, Bifidobacterium infantis,Bifidobacterium adolescentis.
 14. A composition according to any ofclaims 11 to 13, wherein the strain is Lactobacillus acidophilus NCC2463 (CNCM-I 2623), Bifidobacterium bifidum NCC 189 (CNCM-I-2333),Bifidobacterium bifidum NCC 235 (CNCM-I-2335), Bifidobacteriumadolescentis NCC 251 (CNCM-I-2168), Bifidobacterium lactis (ATCC27536).15. A composition according to any of claims 11 to 14, wherein theamount of lactic acid bacteria strain available for the individual iscorresponding to about 10³-10¹⁴ cfu per day.
 16. A composition accordingto any of claims 11 to 15, which reduces, prevents or treats endotoxinmediated and/or associated disorders